Overlooked Enterobacterales as hosts of antimicrobial resistance in aquatic environments

Abstract

The increasing frequency of antibiotic resistant bacteria and their dissemination in environmental microbiomes is a critical health concern. Water quality assessment and AMR surveillance are broadly focused on commonly found Enterobacterales, and mainly on the faecal indicator E. coli. In this study, we analysed antibiotic resistance and biofilm formation in 14 environmental isolates belonging to six neglected species. Genetic diversity was assessed by ERIC-PCR. Identified as Cronobacter sakazakii (1), Kluyvera intermedia (1), Leclercia adecarboxylata (1), Raoultella ornithinolytica (8), Raoultella terrigena (1), and Yersinia massiliensis (2), each isolate had a unique and distinct AMR profile. The isolates demonstrated intrinsic resistance to erythromycin and increased resistance to ampicillin and tetracycline. None of the isolates exhibited carbapenem resistance. Ten isolates were MDR. Thirteen out of the 24 investigated ARGs were detected in bacterial genomes. Except for carbapenemases, various β-lactamases (bla_TEM, bla_CTX-M), and also tet, sul, erm, mef and qnr genes were found. A strong positive correlation was observed between the phenotypic and genotypic resistance. Due to its discriminatory power at the taxonomic level, ERIC-PCR fingerprinting provided a reliable and accurate molecular typing. Negative correlations between the number of ERIC bands, the magnitude of resistance and the biofilm score indicate that strains with abundant ERIC sequences are less likely to be resistant and to adhere to surfaces. This suggests that a high genome plasticity and adaptability prevents specific survival strategies and deserves further attention.

Keywords: Cronobacter; Kluyvera; Leclercia; Raoultella; Yersinia; Biofilm.

Fig. 1

Antibiotic resistance across different species. AMP ampicillin; TZP piperacillin-tazobactam; CAZ ceftazidime; FEP cefepime; IPM imipenem; CIP ciprofloxacin; CN gentamycin; E erythromycin; TE tetracycline; SXT trimethoprim-sulfamethoxazole; R resistant; IR intermediate resistant; n number of isolates.

Fig. 2

ERIC-PCR dendrogram revealing clustering of different Enterobacterales isolates. Green = Raoultella ornithinolytica; dark green = Raoultella terrigena; purple = Leclercia adecarboxylata; fuchsia = Cronobacter sakazakii; yellow = Yersinia massiliensis; pink = Kluyvera intermedia. The grouping of ERIC-PCR profiles includes gel lanes cropped from different parts of the same gel. The full-length gel is included in the Supplementary Data file (Dataset 6). The first and last lanes contain the GeneRuler DNA ladders SM1113 and SM0321 (Thermo Fisher Scientific, USA).

Fig. 3

PCA revealing the variation in the (a) AMR profiles; (b) ARG patterns and (c) ERIC-PCR fingerprints of infrequent Enterobacterales.